Alleviation of ischemia-reperfusion injury in rat liver transplantation by induction of small interference RNA targeting Fas.

ABSTRACT

BACKGROUND: Cellular apoptosis plays an important role in ischemia-reperfusion (I/R) injury during organ transplantation. Synthetic small interference RNA (siRNA) targeting apoptotic receptor Fas has proven effective to protect mice against hepatitis and renal I/R injury. The objective of this study is to investigate the silencing impact of Fas siRNA to alleviate I/R injury in rat liver transplantation. MATERIALS AND METHODS: Rat hepatocytes (BRL cells) were transfected with three pairs of synthesized Fas siRNA; cells untreated and treated with GFP siRNA were taken as blank and siRNA control. The most effective Fas siRNA was chosen for in vivo experiments. Syngeneic orthotopic liver transplantation was performed in Fas siRNA group, siRNA control group, and blank control group of Sprague-Dawley rats. There were 25 pairs of rats in each group. siRNA transfection of donor rats was done with hydrodynamic injection method 48 h before liver procurement. Blood and liver samples were collected for evaluation of serum ALT levels, Fas protein and mRNA expression, and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, 1, 3, 6, 12, and 24 h after liver transplantation. RESULTS: Fas siRNA2, which inhibited Fas gene expression much more than other siRNAs, was chosen for in vivo experiment. The serum ALT levels of Fas siRNA group were much less than those of blank and siRNA control groups 1, 3, 6, 12, and 24 h after blood reperfusion, indicating diminishing ischemia-reperfusion injury. Donor livers in Fas siRNA group had substantially less cell apoptosis. The expression of Fas mRNA and protein was reduced dramatically in the Fas siRNA group compared with the other two groups. CONCLUSION: Fas-mediated apoptosis play an important role in I/R injury of rat liver transplantation. Silencing Fas by hydrodynamic injection of siRNA holds therapeutic promise to limit I/R injury.